Article Figures & Data
Figures
- FIG 1
mRNA analysis of endometrial cancer specimens by real-time RT-PCR. (A and B) Twenty normal endometrial tissue specimens (NEM) and 37 primary HEC specimens were used to analyze their mRNA levels of BHLHE40 (A) and BHLHE41 (B). (C and D) BHLHE40/41 mRNA levels of the HEC group at the early stage (stage IA based on FIGO 2008 system criteria) were compared with those of the group at the advanced stage (more than stage IB). (E) The relationship between BHLHE40 and BHLHE 41 mRNA levels from 37 specimens was analyzed by Pearson's product-moment correlation coefficient. r values are correlation coefficients. The significance of these relationships was determined by the F-test. (F and G) The relationship between TWIST1 and BHLHE40 or TWIST1 and BHLHE41 mRNA levels was also analyzed by Pearson's product-moment correlation coefficient. A P value of <0.05 was considered significant. n.s., not significant.
- FIG 2
Immunohistochemistry of endometrial cancer specimens. Surgical samples from 86 HEC patients were analyzed for BHLHE40/41expression by immunohistochemistry. Representative results are shown. (A to D) Results are for the late proliferative phase (A and B) and the secretory phase (C and D) of normal endometrial tissue. (E and F) A grade 1 endometrioid adenocarcinoma (EAC) case at stage IA; (G and H) a grade 2 EAC case at stage IA; (I and J) a grade 3 EAC case at stage IB; (K and L) a grade 2 EAC case at stage IIIA. Immunohistochemical images with an anti-BHLHE40 antibody (A, C, E, G, I, K) and anti-BHLHE41 antibody (B, D, F, H, J, L) are shown. The scale bars represent 200 μm. (M and N) The staining scores of immunohistochemical images were analyzed. Eighty-six cases were divided into a group at the early stage (stage IA) and that at the advanced stage (more than stage IB). (O) The relationship between BHLHE40 and BHLHE41 staining levels from 86 specimens was analyzed by Pearson's product-moment correlation coefficient. r values are correlation coefficients. The significance of coefficients was determined by the F-test. (P and Q) The relationship between TWIST1 and BHLHE40 or TWIST1 and BHLHE41 staining levels was also analyzed by Pearson's product-moment correlation coefficient. A P value of <0.05 was considered significant.
- FIG 3
Impact of forced BHLHE40/41 expression in HEC cells. (A) Expression status of BHLHE40/41 in HEC cell lines analyzed by immunoblotting. (B) In vitro invasion of Ishikawa, HEC-1, and HEC-6 cells infected with lentivirus vectors to express HA-BHLHE40, FLAG-BHLHE41, or both. The scale bars represent 200 μm. The right graphs show quantification data of the results. (C) Protein expression of EMT markers in Ishikawa, HEC-1, and HEC-6 cells transfected with vectors to express HA-BHLHE40, FLAG-BHLHE41, or both. (D) mRNA levels of SNAI1, SNAI2, and TWIST1 in each transfectant were analyzed by real-time RT-PCR. E40, BHLHE40; E41, BHLHE41; n.s., not significant; P.C., positive control; Lt-Ctrl, control lentivirus vector; *, P < 0.05; **, P < 0.01.
- FIG 4
Impact of BHLHE40/41 in HEC cells analyzed by knockdown. (A) Protein expression levels of EMT markers in HHUA cells infected with lentivirus vectors to knock down BHLHE40, BHLHE41, or both were determined by immunoblotting. (B) A mRNA analysis of SNAI1, SNAI2, and TWIST1 in each knocked-down cell type was performed by real-time RT-PCR. (C) In vitro invasion of each knocked-down cell type. The right graph shows quantification data of the results. The scale bars represent 200 μm. shE40, shBHLHE40; shE41, shBHLHE41; *, P < 0.05; **, P < 0.01.
- FIG 5
Identification of the BHLHE40/41 responsible site in the TWIST1 promoter. (A) Reporter analysis of the SNAI1, SNAI2, and TWIST1 promoters in Ishikawa, HEC-1, and HEC-6 cells transfected with vectors to express HA-BHLHE40 and/or FLAG-BHLHE41. (B) Reporter analysis of the SNAI1, SNAI2, and TWIST1 promoters in HHUA cells transfected with the vectors to knock down BHLHE40 and/or BHLHE41. (C) Truncation assay of TWIST1 reporters. Five kinds of reporters possessing upstream regions from bp −1876, −1587, −170, +34, and +116 from the transcription start site were analyzed for their activity. (D) The pTWIST1+116 reporters possessing a deletion or mutations at the SP1 binding site (SBS) were used to analyze their activity. (E) The control activity of the mutant pTWIST1+116 reporter was adjusted to the same value as that of the wild-type reporter to evaluate the effects of BHLHE40/41 expression (white bars). (F) Impact of the SBS in the full-length TWIST1 reporter was evaluated using the pTWIST1-1876 reporter possessing a mutation at the SBS. The control activity of the mutant pTWIST1-1876 reporter was adjusted to the same value as that of the wild-type reporter to evaluate the effects of BHLHE40/41 expression (white bars). n.s., not significant; *, P < 0.05; **, P < 0.01. #, P < 0.05; ##, P < 0.01 (significantly different from the pCDNA3 control). §, P < 0.05; §§, P < 0.01 (significantly different from the pCDNA3 control).
- FIG 6
SP1 is critical for TWIST1 transcription. (A and B) Immunoblotting analysis (A) and mRNA analysis (B) of EMT marker expression in HEC-1 and HEC-6 cells with SP1 knockdown. (C) HEC-1 and HEC-6 cells showing SP1 knockdown were used for the in vitro cell invasion assay. The graphs show quantification of the data. The scale bars represent 200 μm. (D and E) 293T cells transfected with MYC-SP1 alone or with a combination of MYC-SP1 and HA-BHLHE40 and/or FLAG-BHLHE41 were analyzed for their EMT marker expression at the protein (D) and mRNA (E) levels. (F) The wild-type pTWIST+116 reporter was transfected into HEC-6 cells with MYC-SP1 alone or with a combination of MYC-SP1 and HA-BHLHE40 and/or FLAG-BHLHE41, and its activity was compared with that of control transfectants without MYC-SP1. (G) Same as panel F, but siSP1 was used instead of MYC-SP1. (H) Same as panel F, but the pTWIST+116 reporter possessing mutations at the SBS was used instead of the wild-type reporter. n.s., not significant; *, P < 0.05; **, P < 0.01.
- FIG 7
BHLHE40/41 suppressed SP1 function by a competitive mechanism for binding to the SBS of the TWIST1 promoter. (A) Immunoprecipitation (IP) assay using 293T cells transfected with MYC-SP1, HA-BHLHE40, and FLAG-BHLHE41. The cell lysate was immunoprecipitated with the antibodies shown at the top and immunoblotted with the antibodies indicated on the left (α, anti-). P, cells transfected with pCDNA3 alone; T, cells transfected with all three constructs. (B) IP assay using 293T cells transfected with MYC-SP1, HA-BHLHE40, and FLAG-BHLHE40 (left panels) or 293T cells transfected with MYC-SP1, HA-BHLHE41, and FLAG-BHLHE41 (right panels). The arrowheads indicate the target bands. The bands indicated by asterisks are IgG heavy chains. (C) Electrophoretic mobility shift assay using the nuclear extract of 293T cells transfected with HA-BHLHE40 and FLAG-BHLHE41. Two main SP1-DNA complexes (complexes a and b) were formed. Supershift bands were formed by incubation with anti-HA, -FLAG, and -SP1 antibodies. An anti-CEBPA antibody was used as a negative control. wt, wild type; mt, mutant. (D) Nuclear extracts from 293T cells transfected with HA-BHLHE40 and/or FLAG-BHLHE41 were incubated with the labeled SBS probe. An anti-SP1 antibody was used to form supershift bands. The right graph shows the quantified band intensities of complex b in lanes 1, 3, 5, and 7 from four independent experiments. (E) Chromatin immunoprecipitation assay using 293T cells transfected with HA-BHLHE40 and FLAG-BHLHE41. Protein-DNA complexes immunoprecipitated with each of the anti-SP1, -acetylated H3, -KAT2B, and -HDAC1 antibodies were used to amplify the SBS site by PCR. The 10% input samples were used to calculate the occupancy ratio (%) from the values measured by real-time PCR. **, P < 0.01.
- FIG 8
The SP1 inhibitor WP631 was used to suppress EMT in HEC cells. (A and B) Noncytotoxic concentrations of WP631 were used in HEC-1 (A) and HEC-6 (B) cells in order to examine in vitro cell invasion. The left graphs show cell viability assayed at each concentration of WP631. The right graphs show quantification of the data. The scale bars represent 200 μm. (C) The mRNA levels of TWIST1 in HEC-1 and HEC-6 cells treated with each concentration of WP631 were examined by real-time RT-PCR. (D) Protein levels of EMT markers, including TWIST, in HEC-1 and HEC-6 cells were analyzed by immunoblotting. (E) The wild-type and mutant pTWIST1+116 reporters were used to examine their responses to WP631 in HEC-6 cells. The control activities of the mutant reporter were adjusted to the same values as those of the wild-type reporter and are shown as white bars. (F) Electrophoretic mobility shift assay showing the inhibition of SP1-DNA complexes by WP631 by use of the nuclear extract from 293T cells. An anti-SP1 antibody was used to confirm that the complexes contained SP1 (supershift). n.s., not significant; *, P < 0.05; **, P < 0.01.
Tables
- TABLE 1
Primers used for the qRT-PCR analysis
Target gene Accession no. Sequence Amplicon (bp) Forward primer Reverse primer BHLHE40 NM_003670 5′-GACCGGATTAACGAGTGCAT-3′ 5′-TGCTTTCACATGCTTCAAGG-3′ 123 BHLHE41 NM_030762 5′-GCATGAAACGAGACGACACC-3′ 5′-ATTTCAGATGTTCAGGCAGT-3′ 126 SNAI1 NM_005985 5′-AAGGCCTTCTCTAGGCCCT-3′ 5′-CGCAGGTTGGAGCGGTCAG-3′ 113 SNAI2 NM_003068 5′-TTCGGACCCACACATTACCT-3′ 5′-GCAGTGAGGGCAAGAAAAAG-3′ 122 TWIST1 NM_000474 5′-CAGCTACGCCTTCTCGGTCT-3′ 5′-CTGTCCATTTTCTCCTTCTCTGGA-3′ 138 ACTB NM_001101 5′-TTGCCGACAGGATGCAGAAG-3′ 5′-CAGCGAGGCCAGGATGGAGC-3′ 122 - TABLE 2
Sequences of reporter constructs
Construct Sense oligonucleotidea pTWIST1-wtSP1BS +151 GTCCCCTCCCCCTCCCGCCTCCCTCCCCGCCTCC +184 pTWIST1-mtSP1BS-a +151 GTCCACTACACATCACGCCTCCCTCCCCGCCTCC +184 pTWIST1-mtSP1BS-b +151 GTCCCCTCCCCCTCCCGCATACATACACGACTCC +184 pTWIST1-mtSP1BS-ab +151 GTCCACTACACATCACGCATACATACACGACTCC +184 pTWIST1-wtSP1BS-c +193 CCCTCCCC +200 pTWIST1-mtSP1BS-c +193 ACATACAC +200 ↵a Mutated nucleotides are underlined.
- TABLE 3
Sequences of shRNA constructs
shRNA name Target Target site shRNA sequence (cloned into AgeI and EcoRI)a Control shRNA (shCtrl) None CCTAAGGTTAAGTCGCCCTCG 5′-accggtCCTAAGGTTAAGTCGCCCTCGCTCGAGCGAGGGCGACTTAACCTTAGGTTTTTTTgaattc-3′ shBHLHE40S1 (TRCN0000013249) BHLHE40 exon 4 GCACTAACAAACCTAATTGAT 5′-accggtGCACTAACAAACCTAATTGATCTCGAGATCAATTAGGTTTGTTAGTGCTTTTTTgaattc-3′ shBHLHE40S2 (TRCN0000232187) BHLHE40 exon 4 CATGTGAAAGCACTAACAAAC 5′-accggtGCATGTGAAAGCACTAACAAACCTCGAGGTTTGTTAGTGCTTTCACATGCTTTTTTgaatttc-3′ shBHLHE41S1 (TRCN0000086556) BHLHE41 exon 2 CTGGACTATTCCTCTTTGTAT 5′-accggtGCTGGACTATTCCTCTTTGTATCTCGAGATACAAAGAGGAATAGTCCAGCTTTTTTgaattc-3′ shBHLHE41S2 (TRCN0000016945) BHLHE41 exon 1 AGAGACAGTTACTGGAACATA 5′-accggtAGAGACAGTTACTGGAACATACTCGAGTATGTTCCAGTAACTGTCTCTTTTTTTgaattc-3′ ↵a Cloning sites are shown in lowercase letters.
Supplemental material
- Supplemental file 1 -
Fig. S1 (BHLHE40 and BHLHE41 mRNA and protein levels)
PDF, 46K
- Supplemental file 2 -
Fig. S2 (Protein expression analysis)
PDF, 249K
- Supplemental file 3 -
Fig. S3 (In vitro cell motility)
PDF, 782K
- Supplemental file 4 -
Fig. S4 (mRNA analysis by real-time RT-PCR)
PDF, 37K
- Supplemental file 5 -
Fig. S5 (Band intensities in Fig. 3C and 4A)
PDF, 165K
- Supplemental file 6 -
Fig. S6 (Band intensities in Fig. 6A and D and 8D)
PDF, 172K
- Supplemental file 7 -
Fig. S7 (BHLHE41 and reporter activity of pGL4.22-basic)
PDF, 42K
- Supplemental file 8 -
Legends to Fig. S1 to S7
PDF, 13K
- Supplemental file 1 -
